Abstract

In mammals, hair cells and auditory neurons lack the capacity to regenerate, and damage to either cell type can result in hearing loss. Replacement cells for regeneration could potentially be made by directed differentiation of human embryonic stem (hES) cells. To generate sensory neurons from hES cells, neural progenitors were first made by suspension culture of hES cells in a defined medium. The cells were positive for nestin, a neural progenitor marker, and Pax2, a marker for cranial placodes, and were negative for alpha-fetoprotein, an endoderm marker. The precursor cells could be expanded in vitro in fibroblast growth factor (FGF)-2. Neurons and glial cells were found after differentiation of the neural progenitors by removal of FGF-2, but evaluation of neuronal markers indicated insignificant production of sensory neurons. Addition of bone morphogenetic protein 4 (BMP4) to neural progenitors upon removal of FGF-2, however, induced significant numbers of neurons that were positive for markers associated with cranial placodes and neural crest, the sources of sensory neurons in the embryo. Neuronal processes from hES cell-derived neurons made contacts with hair cells in denervated ex vivo sensory epithelia and expressed synaptic markers, suggesting the formation of synapses. In a gerbil model with a denervated cochlea, the ES cell-derived neurons engrafted in the auditory nerve trunk and sent out neurites that grew toward the auditory sensory epithelium. These data indicate that hES cells can be induced to form sensory neurons that have the potential to treat neural degeneration associated with sensorineural hearing loss.